WO2021235916A1 - 글루카곤, glp-1 및 gip 삼중 활성체의 지속형 결합체의 액상 제제 - Google Patents

글루카곤, glp-1 및 gip 삼중 활성체의 지속형 결합체의 액상 제제 Download PDF

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WO2021235916A1
WO2021235916A1 PCT/KR2021/006462 KR2021006462W WO2021235916A1 WO 2021235916 A1 WO2021235916 A1 WO 2021235916A1 KR 2021006462 W KR2021006462 W KR 2021006462W WO 2021235916 A1 WO2021235916 A1 WO 2021235916A1
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liquid formulation
pro
ser
salts
seq
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PCT/KR2021/006462
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English (en)
French (fr)
Korean (ko)
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임형규
김상윤
배성민
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한미약품 주식회사
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Priority to BR112022023609A priority Critical patent/BR112022023609A2/pt
Priority to EP21809116.3A priority patent/EP4154870A1/en
Priority to US17/926,912 priority patent/US20230285583A1/en
Priority to CA3179649A priority patent/CA3179649A1/en
Priority to CN202180048037.XA priority patent/CN115811970A/zh
Priority to JP2022571835A priority patent/JP2023526552A/ja
Priority to AU2021274518A priority patent/AU2021274518A1/en
Priority to IL298415A priority patent/IL298415A/en
Priority to MX2022014666A priority patent/MX2022014666A/es
Publication of WO2021235916A1 publication Critical patent/WO2021235916A1/ko

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    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

  • the present invention relates to a liquid formulation of a long-acting conjugate of glucagon, GLP-1 and a triple activator of GIP, and a method for preparing the same.
  • Diabetes including obesity and type 2 diabetes, is one of the representative metabolic diseases occurring in modern society and is recognized as a global health threat, and the economic costs accompanying the onset of the disease are also on the rise.
  • GLP-1 and GIP are representative gastrointestinal hormones and neurohormones that are involved in regulating blood sugar levels following food intake.
  • Glucagon is a peptide hormone secreted by the pancreas and is involved in blood sugar concentration control together with the above two substances.
  • GLP-1 is a hormone secreted from the small intestine when stimulated by food intake. It promotes insulin secretion from the pancreas in a blood sugar concentration-dependent manner and suppresses the secretion of glucagon to help lower blood sugar levels. In addition, it acts as a satiety factor, slowing the digestive action of the stomach and delaying the passage time of the digested food, thereby reducing food intake. Moreover, when administered to mice, it was reported that food intake suppression and weight loss effects were reported, and these effects were confirmed to be the same in both normal and obese states, showing potential as a treatment for obesity.
  • GIP GIP-1
  • GIP one of the gastrointestinal hormones secreted when stimulated by food intake, is a hormone composed of 42 amino acids secreted from K cells in the small intestine. It performs a function that helps to lower it, and the effect of increasing the activity of GLP-1 and the anti-inflammatory effect have been reported.
  • Glucagon is produced by the pancreas when blood sugar begins to drop due to reasons such as medication or disease, hormone or enzyme deficiency. Glucagon is responsible for signaling the liver to break down glycogen to release glucose and raise blood sugar levels to normal levels. In addition, glucagon has an anti-obesity effect by promoting lipolysis and energy expenditure by activating hormone sensitive lipase in adipocytes and suppressing appetite in animals and humans, in addition to the effect of raising blood sugar. has been reported to appear.
  • GLP-1, GIP, and glucagon receptors are being developed.
  • substances with various activation ratios for GLP-1, GIP, and glucagon receptors for example, have high activity of GLP-1 and GIP for enhancing blood glucose and relatively low activity of glucagon, which has a weight loss effect, but has a greater glycemic control ability.
  • a substance having a high weight loss effect is being developed due to a high substance, but high activity of GLP-1, GIP, and glucagon.
  • the present invention provides a liquid formulation of a long-acting conjugate of glucagon, GLP-1 and a triple activator of GIP.
  • Another object of the present invention is to provide a method for preparing the liquid formulation.
  • the liquid formulation according to the present invention has the advantage of economical provision by providing storage stability to the binder of the present invention having a large molecular weight with a simple formulation.
  • 1A and 1B show the long-acting forms of glucagon, GLP-1 and GIP triple activators according to the type of buffer material based on the liquid formulation of Example 3 (sodium citrate, pH 5.5, mannitol, polysorbate 20, methionine) This is the result of confirming the stability of the conjugate.
  • the composition shown in Table 9 was used as a liquid formulation of a long-acting conjugate of glucagon, GLP-1 and GIP triple activator, respectively, and the stability results were shown after storage at 25° C. for 6 weeks.
  • One embodiment embodying the present invention is a liquid formulation of a long-acting conjugate of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) triple activator.
  • the long-acting conjugate refers to a substance in which a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor is covalently bonded to an immunoglobulin Fc fragment by a linker.
  • the present invention relates to a glucagon receptor, a GLP-1 receptor, and a GIP receptor, wherein a peptide and an immunoglobulin Fc fragment having activity on the glucagon receptor, the GLP-1 receptor, and the GIP receptor are linked to each other.
  • a glucagon receptor, GLP-1 receptor comprising a pharmacologically effective amount of a long-acting conjugate of a peptide having , and to a liquid formulation of a long-acting conjugate of a peptide having activity against a GIP receptor.
  • the long-acting conjugate refers to a substance in which a peptide having activity for a glucagon receptor, a GLP-1 receptor, and a GIP receptor is covalently bound to an immunoglobulin Fc fragment.
  • the liquid formulation may include a long-acting conjugate of Formula 1; buffer substances; And it is characterized in that it is a liquid formulation of a long-acting conjugate comprising a sugar alcohol, sugar, or a combination thereof:
  • Q is a peptide of Formula 1 below;
  • L is a linker containing ethylene glycol repeating units
  • a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
  • Z is an immunoglobulin Fc fragment
  • a lactam ring is formed between the glutamic acid (Glu) at position 16 and the lysine (Lys) residue at position 20 from the underlined N-terminus,
  • Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine,
  • Xaa3 is glutamic acid or glutamine
  • Xaa10 is tyrosine or cysteine
  • Xaa12 is lysine or isoleucine
  • Xaa13 is tyrosine, alanine, or cysteine
  • Xaa14 is leucine or methionine
  • Xaa15 is cysteine or aspartic acid
  • Xaa17 is arginine, isoleucine, cysteine, or lysine,
  • Xaa18 is alanine, arginine, or histidine
  • Xaa19 is alanine, glutamine, or cysteine
  • Xaa21 is glutamic acid or aspartic acid
  • Xaa24 is glutamine, asparagine, or aspartic acid
  • Xaa28 is alanine, asparagine, or aspartic acid
  • Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine;
  • Xaa30 is cysteine, glycine, lysine, or histidine, or is absent;
  • R1 is cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n ( SEQ ID NO: 49) or absent,
  • n is Cys, or Pro
  • n is Cys, or Gly, or may be absent.
  • the Aib means aminoisobutyric acid.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation contains 18 to 920 nmol/mL of the long-acting conjugate of Formula 1; a buffer material in an amount for maintaining the pH of the liquid formulation in the range of 5.0 to 7.0; and 0.5 to 10% (w/v) of sugar alcohol, sugar, or a combination thereof; characterized in that it is a liquid formulation of a long-acting binder.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation further comprises one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation does not contain an isotonic agent.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation does not additionally include one or more components selected from the group consisting of a nonionic surfactant and amino acids.
  • liquid formulation according to any one of the preceding embodiments, wherein the peptide comprises any one amino acid sequence selected from SEQ ID NOs: 1 to 46.
  • liquid formulation according to any one of the preceding embodiments, wherein the peptide comprises any one amino acid sequence selected from SEQ ID NOs: 1, 2, 9, 19, 21 to 27, 30 to 32, or 40 to 46 do.
  • liquid formulation according to any one of the preceding embodiments, wherein the peptide comprises any one amino acid sequence selected from SEQ ID NOs: 9. 30 to 32, or 42 to 46.
  • liquid formulation according to any one of the preceding embodiments, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 9.
  • R1 is cysteine, Cys-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 50), Pro-Ser-Ser-Gly-Ala- Pro-Pro-Pro-Ser (SEQ ID NO: 51), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly (SEQ ID NO: 52), Pro-Ser-Ser-Gly-Gln-Pro- Pro-Pro-Ser (SEQ ID NO: 53), or Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 54), or absent.
  • liquid formulation according to any one of the preceding embodiments, wherein L is polyethylene glycol.
  • liquid formulation according to any one of the preceding embodiments, wherein the formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.
  • liquid formulation according to any one of the preceding embodiments, wherein the ethylene glycol repeating unit is [OCH 2 CH 2 ]n, n is a natural number, and the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, such as the number It is characterized in that the average molecular weight is determined to be 1 to 100 kDa.
  • n is determined such that the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, for example, the number average molecular weight is 10 kDa.
  • liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is derived from IgG4.
  • liquid formulation according to any one of the preceding embodiments, characterized in that the immunoglobulin Fc fragments Z and Q are not glycosylated.
  • the buffer material is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof.
  • the buffer material is selected from the group consisting of a citrate buffer solution, an acetate buffer solution, a histidine buffer solution, and combinations thereof.
  • liquid formulation according to any one of the preceding embodiments, wherein the buffer material is acetic acid and a salt thereof.
  • liquid formulation according to any one of the preceding embodiments, wherein the pH of the liquid formulation is 5.0 to 5.5.
  • liquid formulation according to any one of the preceding embodiments, wherein the pH of the liquid formulation is 5.0 to 6.5.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation has a pH of 5.1 to 6.0.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation has a pH of 5.1 to 5.5.
  • liquid formulation according to any one of the preceding embodiments, wherein the sugar or sugar alcohol is at least one selected from the group consisting of sucrose, mannitol, and sorbitol.
  • liquid formulation according to any one of the preceding embodiments, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.
  • liquid formulation according to any one of the preceding embodiments, wherein the sugar is sucrose.
  • liquid formulation according to any one of the preceding embodiments, wherein the sugar alcohol is at least one selected from the group consisting of mannitol and sorbitol.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation further comprises one or more components selected from the group consisting of nonionic surfactants and amino acids.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation further comprises an isotonic agent.
  • liquid formulation according to any one of the preceding embodiments, characterized in that it contains 0.01 to 0.1% (w/v) of a nonionic surfactant.
  • liquid formulation according to any one of the preceding embodiments, wherein the nonionic surfactant is a poloxamer, a polysorbate, or a combination thereof.
  • nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. characterized in that it is selected.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation further comprises an amino acid selected from the group consisting of arginine, glycine, methionine, and combinations thereof as a stabilizer.
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 6.5; 1 to 10% (w/v) of a sugar alcohol, sugar, or a combination thereof; And 0.01 ⁇ 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and 0.01 ⁇ 1 mg / selected from the group consisting of arginine, glycine, methionine, and combinations thereof mL of a stabilizing agent.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 6.5
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; And 0.01 ⁇ 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and 0.01 ⁇ 1 mg / selected from the group consisting of arginine, glycine, methionine, and combinations thereof mL of a stabilizing agent.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; 4-10% (w/v) sugar; and 0.01 to 1 mg/mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; and 4 to 10% (w/v) of sugar.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; and 4 to 10% (w/v) of sugar.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w/v) of sugar.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w/v) of sugar.
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; and 0.01 to 1 mg/mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; and 0.01 to 0.1% (w/v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • liquid formulation contains 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; a stabilizer of 0.01 to 1 mg/mL selected from the group consisting of arginine, glycine, methionine, and combinations thereof; and 0.01 to 0.1% (w/v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • a stabilizer of 0.01 to 1 mg/mL selected from the group consisting of arginine, glycine, methi
  • liquid formulation according to any one of the preceding embodiments, wherein the amino acid is methionine.
  • liquid formulation according to any one of the preceding embodiments, wherein the methionine is present in a concentration of 0.01 to 1 mg/mL in the formulation.
  • liquid formulation according to any one of the preceding embodiments, wherein the amino acid is methionine or arginine.
  • liquid formulation according to any one of the preceding embodiments, wherein the methionine or arginine is present in the formulation at a concentration of 0.01 to 1 mg/mL.
  • liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation is characterized in that it is transparent in appearance when stored for one week under conditions of 40 ⁇ 2 °C and 75 ⁇ 5% of relative humidity, which are severe test conditions.
  • immunoglobulin Fc fragment is an Fc fragment derived from IgG, IgA, IgD, IgE or IgM.
  • the immunoglobulin Fc fragment comprises (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or more domains of a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain with an immunoglobulin hinge region or portion of a hinge region; And (f) it is characterized in that it is selected from the group consisting of a dimer of each domain of the heavy chain constant region and the light chain constant region.
  • each domain of the immunoglobulin Fc fragment comprises a domain of different origin derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. It is characterized as a hybrid.
  • liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is in the form of a dimer or multimer composed of a single-chain immunoglobulin composed of domains of the same origin.
  • liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is an IgG4 Fc fragment.
  • liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is a human aglycosylated IgG4 Fc fragment.
  • liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is modified to remove a site capable of forming a disulfide bond, modified to remove some N-terminal amino acids from native Fc, Derivatives of native Fc, including a modification in which a methionine residue is added to the N-terminus, a modification in which the complement binding site is removed, or a modification in which an antibody dependent cell mediated cytotoxicity (ADCC) site is removed, or a combination of the above modifications characterized by being.
  • ADCC antibody dependent cell mediated cytotoxicity
  • Another embodiment of the present invention is a method for preparing the liquid formulation.
  • the present invention relates to (a) a glucagon receptor, a GLP-1 receptor, and a GIP receptor, in which a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor and an immunoglobulin Fc fragment are linked to each other.
  • a liquid formulation of a long-acting conjugate which is the liquid formulation according to any one of the preceding embodiments, comprising mixing (b) i) a buffer substance and ii) sugar or sugar alcohol with the long-acting conjugate of a peptide having activity against It relates to a manufacturing method.
  • liquid formulation further comprises one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.
  • One embodiment embodying the present invention provides a liquid formulation of a long-acting conjugate of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (glucose-dependent insuliontropic polypeptide) triple activator.
  • the present invention relates to a glucagon receptor, a GLP-1 receptor, and a peptide having activity on the GIP receptor, and an immunoglobulin Fc fragment linked to each other, a glucagon receptor, a GLP-1 receptor, and a peptide having activity on the GIP receptor.
  • the present invention relates to a long-acting conjugate of Formula 1; buffer substances; and a sugar alcohol, sugar, or a combination thereof; provides a liquid formulation comprising:
  • Q is a peptide of Formula 1 below;
  • L is a linker containing ethylene glycol repeating units
  • a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
  • Z is an immunoglobulin Fc fragment
  • a lactam ring is formed between the glutamic acid (Glu) at position 16 and the lysine (Lys) residue at position 20 from the underlined N-terminus,
  • Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine,
  • Xaa3 is glutamic acid or glutamine
  • Xaa10 is tyrosine or cysteine
  • Xaa12 is lysine or isoleucine
  • Xaa13 is tyrosine, alanine, or cysteine
  • Xaa14 is leucine or methionine
  • Xaa15 is cysteine or aspartic acid
  • Xaa17 is arginine, isoleucine, cysteine, or lysine,
  • Xaa18 is alanine, arginine, or histidine
  • Xaa19 is alanine, glutamine, or cysteine
  • Xaa21 is glutamic acid or aspartic acid
  • Xaa24 is glutamine, asparagine, or aspartic acid
  • Xaa28 is alanine, asparagine, or aspartic acid
  • Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine;
  • Xaa30 is cysteine, glycine, lysine, or histidine, or is absent;
  • R1 is cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n ( SEQ ID NO: 49) or absent,
  • n is Cys, or Pro
  • n is Cys, or Gly, or absent.
  • liquid preparation refers to a drug formulated in a liquid form, and includes both liquid internal preparations and external preparations.
  • the liquid formulation of the present invention includes the long-acting conjugate of Formula 1 exhibiting a pharmacological effect and a substance capable of stably maintaining and/or storing the substance exhibiting the pharmacological effect for a certain period of time when it is formulated in a liquid form.
  • Components included in addition to the long-acting conjugate of Formula 1 that exhibits the pharmacological effect of the liquid formulation may be mixed with the stabilizer.
  • liquid formulation of the long-acting conjugate of Formula 1 of the present invention storage stability is important to ensure accurate dosage.
  • the present invention relates to a specific concentration of a long-acting conjugate of Formula 1, which is a substance exhibiting a pharmacological effect; an amount of buffer material to maintain the pH in the range of 5.0 to 7.0; and 0.5 to 10% (w/v) of sugar alcohol, sugar, or a combination thereof; to provide a new formulation of the present invention by confirming that it is stable even during long-term storage of the long-acting conjugate of Formula 1 became
  • the concentration of the long-acting conjugate of the peptide having activity on the glucagon receptor, GLP-1 receptor, and GIP receptor contained in the liquid formulation of the present invention may be 18 nmol/mL to 1840 nmol/mL, but is not limited thereto. .
  • the concentration of the long-acting conjugate may be 18 to 920 nmol/mL, but is not limited thereto.
  • the term "stabilizer” refers to a substance that stably maintains components such as active ingredients in a formulation for a certain period of time.
  • the stabilizer of the present invention preferably does not contain albumin.
  • Human serum albumin which can be used as a protein stabilizer, is manufactured from human blood, so there is a possibility of contamination by a human-derived pathogenic virus, and gelatin or bovine serum albumin may cause disease or allergic reactions in some patients. have.
  • the albumin-free stabilizer of the present invention does not contain heterologous proteins such as human or animal-derived serum albumin or purified gelatin, so there is little risk of viral infection.
  • the stabilizing agent refers to a substance that enables stable storage of a long-acting conjugate of glucagon, GLP-1, and a triple activator of GIP in particular.
  • storage stability is not only important to ensure correct dosage, but also potential generation of antigenic substances against glucagon, GLP-1 and GIP triple activator conjugates. important to suppress
  • the buffer material which is one component included in the liquid formulation of the present invention, can maintain the pH of the solution so that the pH of the liquid formulation does not change rapidly so that the long-acting conjugate of Formula 1 is stable.
  • the buffer material may also be referred to as a buffer system, and the buffer material or buffer system serves to maintain the pH of the liquid formulation.
  • a buffer material capable of maintaining a pH capable of stabilizing the long-acting conjugate of Formula 1, which is a target material to be stabilized, may be used without limitation.
  • the buffer material is phosphoric acid and its conjugate salt, an alkali salt (eg, phosphate: sodium phosphate, potassium phosphate or a hydrogen or dihydrogen salt thereof), citric acid and a salt thereof (eg sodium citrate), acetic acid and its salt (eg, sodium acetate), and a pH buffer including histidine and salts thereof, and mixtures of these buffers may also be used, but are not limited thereto.
  • an alkali salt eg, phosphate: sodium phosphate, potassium phosphate or a hydrogen or dihydrogen salt thereof
  • citric acid and a salt thereof eg sodium citrate
  • acetic acid and its salt eg, sodium acetate
  • a pH buffer including histidine and salts thereof e.g, sodium acetate
  • the liquid formulation of the present invention may include a buffer solution containing the buffer material as a solvent of the liquid formulation, specifically, the buffer solution is a citrate buffer solution (eg, sodium citrate buffer), an acetate buffer solution (eg, It may be selected from the group consisting of sodium acetate buffer solution), phosphate buffer solution (eg sodium phosphate buffer solution), histidine buffer solution, and combinations thereof, and a buffer material (citric acid and its salt, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, or a combination thereof) may be included in a concentration sufficient to maintain the desired pH of the liquid formulation.
  • a citrate buffer solution eg, sodium citrate buffer
  • an acetate buffer solution eg, It may be selected from the group consisting of sodium acetate buffer solution
  • phosphate buffer solution eg sodium phosphate buffer solution
  • histidine buffer solution eg sodium phosphate buffer solution
  • a buffer material citric acid and its salt
  • the pH of the liquid formulation is from about 5.0 to about pH 7.0, such as from about 5.0 to about pH 6.8, from about 5.0 to about 6.7, from about 5.0 to about 6.6, from about 5.0 to about pH 6.5, from about 5.0 to about 6.4.
  • the concentration of the liquid formulation to achieve the target pH may be about 1 mM to about 200 mM, and more specifically, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 5 mM to about 40 mM, about 8 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 25 mM, about 10 mM to about 25 mM, about 15 mM to about 25 mM, about 18 mM to about 24 mM, about 18 mM to about 22 mM, or about 20 mM, but is not particularly limited thereto.
  • the buffer material may be acetic acid and a salt thereof, but is not limited thereto.
  • the buffer may be an acetate buffer (eg, sodium acetate buffer) or a citrate buffer (eg, sodium citrate buffer), but is not particularly limited thereto.
  • acetate buffer eg, sodium acetate buffer
  • a citrate buffer eg, sodium citrate buffer
  • the components are dissolved in water (eg, WFI), and the pH of the buffer or formulation can be adjusted to a desired pH using HCl and/or NaOH, etc., which is already conventional in the art. method used. Therefore, even if there is no separate reference to the pH adjuster in the claims, it will be understood by those skilled in the art that the formulation can have an adjusted pH through this method.
  • WFI water
  • the pH of the buffer or formulation can be adjusted to a desired pH using HCl and/or NaOH, etc., which is already conventional in the art. method used. Therefore, even if there is no separate reference to the pH adjuster in the claims, it will be understood by those skilled in the art that the formulation can have an adjusted pH through this method.
  • Sugar alcohol which is one component included in the stabilizer of the present invention, refers to a substance containing a plurality of hydroxyl groups, includes a substance in which an aldehyde group and/or a ketone group of a sugar is substituted with an alcohol group, and saccharides containing multiple hydroxyl groups.
  • the sugar or sugar alcohol may increase the stability of the long-acting conjugate of glucagon, GLP-1, and the triple activator of GIP.
  • the sugar alcohol may be one or more selected from the group consisting of mannitol and sorbitol, but is not limited thereto.
  • saccharide refers to monosaccharides, disaccharides, polysaccharides, oligosaccharides, etc., and is a long-acting conjugate of peptides having activity on glucagon receptors, GLP-1 receptors, and GIP receptors.
  • monosaccharides such as mannose, glucose, fructose, galactose, fucose and xylose
  • disaccharides such as lactose, maltose, and sucrose
  • polysaccharides such as raffinose and dextran, but is not limited thereto.
  • the sugar may be glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof, but is not limited thereto.
  • the sugar may be sucrose, but is not particularly limited thereto.
  • the sugar alcohol, sugar, or a combination thereof is about 0.5 to about 20% (w/v), about 0.5 to about 10% (w/v), about 0.5 to about 6% (w/v) of the total solution of the liquid formulation.
  • v about 1 to about 20% (w/v), about 1 to 15% (w/v), about 2 to about 15% (w/v), about 2 to about 12% (w/v), about 2 to about 12% (w/v), about 3 to about 10% (w/v), about 4 to about 10% (w/v), about 4 to about 6% (w/v), about 5 to about 10% (w/v), about 6 to about 10% (w/v), about 7 to about 10% (w/v), about 7 to about 9% (w/v), about 8 to about 9% (w/v), or about 1.0 % (w/v), about 3.0 % (w/v), about 5.0 % (w/v), or about 8.0 % (w/v).
  • it is not particularly limited thereto.
  • liquid formulation may further include one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.
  • the stabilizer of the liquid formulation may consist essentially of i) a buffer material and ii) a sugar or sugar alcohol, but i) a buffer material, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) an amino acid; and iv) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an amino acid; i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent;
  • the nonionic surfactant which is one component included in the liquid formulation, can prevent protein adsorption or aggregation on the hydrophobic surface by lowering the surface tension of the protein solution.
  • nonionic surfactant examples include polysorbates (eg, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) Sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate); (20) means the total number of oxyethylene groups (-(CH2CH2O)-), poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly(propylene oxide)), Polyethylene-polypropylene glycol, polyoxyethylene compounds (such as polyoxyethylene-stearate, polyoxyethylene alkyl ethers (alkyl: C1-C30), polyoxyethylene monoallyl ethers, alkylphenyl polyoxyethylene copolymers (alkyl: C1-C30), etc.), sodium dodecyl
  • the nonionic surfactant may be polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or poloxamer 188, and these may be used in combination, but is not particularly limited thereto. .
  • the nonionic surfactant is not included in a high concentration, and specifically, the concentration of about 0.2% (w/v) or less in the formulation of the present invention, for example, about 0.001 to about 0.2% (w/v) , about 0.001 to about 0.1% (w/v), about 0.001 to about 0.05% (w/v), about 0.005 to about 0.08% (w/v), about 0.002 to about 0.05% (w/v), about 0.005 to about 0.05% (w/v), about 0.01 to about 0.05% (w/v), about 0.01 to about 0.04% (w/v), about 0.01 to about 0.03% (w/v), about 0.01 to It may be included in about 0.1% (w/v), or about 0.02% (w/v), but is not particularly limited thereto.
  • the amino acid which is a kind of stabilizer as an optional component that may be added to the liquid formulation, may be methionine, arginine, glycine, or a combination thereof, but is not limited thereto.
  • the amino acid may be in the L-form, but is not particularly limited thereto.
  • the amino acid may be methionine or arginine.
  • the methionine may be L-methionine
  • the arginine may be L-arginine, but is not particularly limited thereto.
  • the amino acid may inhibit the generation of impurities that may occur due to the oxidation reaction of the protein, but is not particularly limited thereto.
  • the amino acid may be present in the formulation at a concentration of about 0.01 to about 1 mg/mL, about 0.01 to about 0.8 mg/mL, about 0.01 to about 0.5 mg/mL, about 0.02 to about 0.5 mg/mL, or about 0.02 to about 0.4 mg. It may be present in /mL, or about 0.1 mg/mL, but is not particularly limited thereto.
  • the liquid formulation comprising a buffer material and sugar or sugar alcohol, with or without an isotonic agent, a nonionic surfactant, and one or more components selected from the group consisting of amino acids additionally It may not include, but is not limited to.
  • the isotonic agent refers to a substance capable of exhibiting osmotic pressure control.
  • the isotonic agent may serve to properly maintain osmotic pressure when the liquid formulation according to the present invention is administered to the body.
  • isotonic agents include sodium chloride, sodium sulfate, or sodium citrate as a water-soluble inorganic salt, specifically sodium chloride, but is not particularly limited thereto.
  • an inorganic salt may be an optional component further included in the above-described stabilizer, and is not particularly limited thereto.
  • the above-described stabilizing agent may serve as an isotonic agent.
  • the concentration of the isotonic agent in the formulation according to the present invention is 0 to about 200 mM, 0 to about 150 mM, 0 to about 100 mM, about 10 to about 200 mM, about 10 to about 150 mM, about 10 to about 100 mM, about 10 to about 50 mM , about 20 to about 100 mM, about 40 to about 110 mM, about 20 to about 80 mM, about 20 to about 50 mM, about 20 to about 30 mM, or about 40 to about 50 mM, about 40 to about 60 mM, about 90 to about 110 mM
  • the liquid formulation of the present invention includes sugar alcohol, sugar, or a combination thereof, which are essential components of the above-described liquid formulation; And in addition to the buffer material, other components or materials known in the art may be optionally further included in the range that does not impair the effects of the present invention in addition to the nonionic surfactant and amino acids, which are optional components, but is not limited thereto.
  • the formulation may further include a polyhydric alcohol, but is not particularly limited thereto.
  • a buffer material and ii) a sugar or sugar alcohol, as well as a polyhydric alcohol i) a buffer material, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) an amino acid; and iv) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an amino acid; i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent; i) a buffer,
  • polyhydric alcohols examples include propylene glycol and low molecular weight polyethylene glycol, glycerol, low molecular weight polypropylene glycol, and the like, and may be used in one or two or more combinations thereof. , but not limited thereto.
  • the long-acting conjugate of Formula 1 is an active ingredient included in the liquid formulation of the present invention, and may be included in the formulation in a pharmaceutically effective amount.
  • the concentration of the long-acting binder may be about 18 to about 2800 nmol/mL, about 18 to about 2757 nmol/mL, about 18 to about 2576 nmol/mL, about 18 to about 2392 nmol/mL, about 18 to about in the formulation.
  • the term “about” includes all ranges including ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, ⁇ 0.01, etc. However, the present invention is not limited thereto.
  • the liquid formulation may include, in one embodiment, the peptide conjugate of Formula 1; a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the liquid formulation has a pH of 5.0 to 5.5; Party; and a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 6.5; 1 to 10% (w/v) of a sugar alcohol, sugar, or a combination thereof; And 0.01 ⁇ 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and 0.01 ⁇ 1 mg / selected from the group consisting of arginine, glycine, methionine, and combinations thereof It may be a liquid formulation containing mL of a stabilizer.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; And 0.01 ⁇ 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and 0.01 ⁇ 1 mg / selected from the group consisting of arginine, glycine, methionine, and combinations thereof It may be a liquid formulation containing mL of a stabilizer.
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; 4-10% (w/v) sugar; and 0.01 to 1 mg/mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; and 4 to 10% (w/v) of sugar.
  • a buffer material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.1 to 5.5; and 4 to 10% (w/v) of sugar.
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w/v) of sugar.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w/v) of sugar.
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; and 0.01 to 1 mg/mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; and 0.01 to 0.1% (w/v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.
  • a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; and 0.01 to 0.1% (w/v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.
  • the liquid formulation may include, in one embodiment, 90 to 552 nmol/mL of the peptide conjugate of Formula 1; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof so that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w/v) sugar; a stabilizer of 0.01 to 1 mg/mL selected from the group consisting of arginine, glycine, methionine, and combinations thereof; and 0.01 to 0.1% (w/v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.
  • the liquid formulation may have a transparent appearance when stored for one week under harsh test conditions of 40 ⁇ 2 °C and relative humidity of 75 ⁇ 5%.
  • stress testing refers to a test intended to identify the fundamental characteristics of the stability of a drug. It is performed during drug development and is conducted under harsher conditions than accelerated tests, and it helps to identify expected degradation products and physical changes of the drug.
  • the term "long-acting conjugate of a peptide having activity on glucagon receptor, GLP-1 receptor, and GIP receptor” is an active ingredient included in the liquid formulation of the present invention, and may be included in the formulation in a pharmacologically effective amount.
  • the long-acting conjugate may be in a form in which an immunoglobulin Fc fragment is linked to a glucagon receptor, a GLP-1 receptor, and a peptide having activity on the GIP receptor.
  • the conjugate may exhibit increased potency persistence compared to the peptide to which the immunoglobulin Fc fragment is not bound.
  • Long-acting triple-activator conjugate “peptide conjugate” or “long-acting conjugate of Formula 1" may be used interchangeably.
  • the immunoglobulin Fc fragment and Q may not be glycosylated, but is not limited thereto.
  • the combination may be non-naturally occurring.
  • the long-acting conjugate of the present application may be a form in which a peptide having activity on glucagon receptor, GLP-1 receptor, and GIP receptor and an immunoglobulin Fc fragment are linked to each other, and the linking method is not particularly limited, but the peptide through a linker and immunoglobulin Fc fragments may be linked to each other.
  • the long-acting conjugate of the present invention has the structure of Formula 1 below.
  • Q is a peptide of Formula 1 below;
  • L is a linker containing ethylene glycol repeating units
  • a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other;
  • Z is an immunoglobulin Fc fragment
  • Q of the long-acting conjugate of Formula 1 may be a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor.
  • a glucagon receptor a GLP-1 receptor
  • GIP receptor a GIP receptor
  • a peptide having a significant level of activity for the glucagon receptor, the GLP-1 receptor and the GIP receptor may be used interchangeably herein as a "triple activator”.
  • Q of Formula 1 is a glucagon receptor, a GLP-1 receptor and a peptide having activity on a GIP receptor, including the sequence of Formula 1 below, is active on a glucagon receptor, a GLP-1 receptor and a GIP receptor is a peptide having:
  • a lactam ring is formed between the glutamic acid (Glu) at position 16 and the lysine (Lys) residue at position 20 from the underlined N-terminus,
  • Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine,
  • Xaa3 is glutamic acid or glutamine
  • Xaa10 is tyrosine or cysteine
  • Xaa12 is lysine or isoleucine
  • Xaa13 is tyrosine, alanine, or cysteine
  • Xaa14 is leucine or methionine
  • Xaa15 is cysteine or aspartic acid
  • Xaa17 is arginine, isoleucine, cysteine, or lysine,
  • Xaa18 is alanine, arginine, or histidine
  • Xaa19 is alanine, glutamine, or cysteine
  • Xaa21 is glutamic acid or aspartic acid
  • Xaa24 is glutamine, asparagine, or aspartic acid
  • Xaa28 is alanine, asparagine, or aspartic acid
  • Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine;
  • Xaa30 is cysteine, glycine, lysine, or histidine, or is absent;
  • R1 is cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n ( SEQ ID NO: 49) or absent,
  • n is Cys, or Pro
  • n is Cys, or Gly, or absent.
  • Aib of Formula 1 means aminoisobutyric acid.
  • Aib may be used interchangeably with “2-aminoisobutyric acid” or “aminoisobutyric acid”, and 2-aminoisobutyric acid and aminoiso Butyric acid (aminoisobutyric acid) may be used in combination.
  • the peptide may include an amino acid sequence selected from SEQ ID NOs: 1 to 46 and (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 46, but is not limited thereto.
  • a peptide examples include a peptide comprising or (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 9, 19, 21 to 27, 30 to 32, or 40 to 46. , but is not particularly limited thereto.
  • the peptide may be a peptide comprising or (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 9. 30 to 32, or 42 to 46, or the amino acid sequence of SEQ ID NO: 9 It may be a peptide comprising or (essentially) consisting thereof, but is not particularly limited thereto.
  • R1 is cysteine, Cys-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 50), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- Ser (SEQ ID NO: 51), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly (SEQ ID NO: 52), Pro-Ser-Ser-Gly-Gln-Pro-Pro-Ser ( SEQ ID NO: 53), or Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 54), or may be absent, but is not particularly limited thereto.
  • the peptide having activity on the glucagon receptor, the GLP-1 receptor, and the GIP receptor is characterized in that it includes an intramolecular bridge.
  • it may be a covalent cross-linked or a non-covalent cross-linked, and specifically may be in a form including a ring. It may form a ring between glutamic acid, which is amino acid 16, which is the underlined amino acid residue in Formula 1, and lysine, which is amino acid 20, but is not particularly limited thereto.
  • Non-limiting examples of the ring may include a lactam bridge (or lactam ring).
  • the peptide according to the present invention includes all forms of the peptide itself, a salt thereof (eg, a pharmaceutically acceptable salt of the peptide), or a solvate thereof.
  • the peptide may be in any pharmaceutically acceptable form.
  • the type of the salt is not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.
  • pharmaceutically acceptable means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of medical judgment.
  • salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like.
  • Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • solvate refers to a compound in which the peptide or a salt thereof according to the present invention forms a complex with a solvent molecule.
  • such a peptide may be non-naturally occurring.
  • the C-terminus of the peptide may be an amidated peptide or a peptide having a free carboxyl group (-COOH), or may include a peptide having an unmodified C-terminus, but is not limited thereto.
  • Q may have an amidated C-terminus, but is not limited thereto.
  • the Q may be non-glycosylated, but is not limited thereto.
  • the peptide of the general formula 1 may be synthesized through a solid phase synthesis method, may be produced by a recombinant method, and may be produced by requesting commercially, but is not limited thereto.
  • the term "long-acting conjugate of Formula 1" is an active ingredient included in the liquid formulation of the present invention, and may be included in the liquid formulation in a pharmacologically effective amount.
  • the glucagon receptor, the GLP-1 receptor and the peptide having activity on the GIP receptor and the immunoglobulin Fc region are linked to each other by a linker, and the conjugate is a glucagon receptor, GLP-1 to which the immunoglobulin Fc region is not bound. It can exhibit increased potency persistence compared to peptides having activity on receptors and GIP receptors.
  • connection between Q, a peptide having activity on glucagon receptors, GLP-1 receptors and GIP receptors, and the immunoglobulin Fc fragment may be a physical or chemical bond, non-covalent or covalent bond, Specifically, it may be a covalent bond, but is not limited thereto.
  • the method for linking Q which is a peptide having activity to glucagon receptor, GLP-1 receptor, and GIP receptor, and the immunoglobulin Fc fragment is not particularly limited, but via a linker, glucagon receptor, GLP-1 receptor and a peptide having activity against a GIP receptor and an immunoglobulin Fc fragment may be linked to each other.
  • the long-acting binder included in the liquid formulation of the present application may be one represented by Formula 1 above.
  • Q and Z may be bonded to each other through L by a covalent bond.
  • Q and L, and L and Z may be connected to each other by a covalent bond, and in this case, the conjugate may be a conjugate in which Q, L, and Z are each connected through a covalent bond in the order of Formula 1 .
  • Q may be directly linked to Z (ie, a is 0 in Formula 1) or linked through a linker (L).
  • La which is a component of the long-acting conjugate of Formula 1
  • L which is a linker including the ethylene glycol repeating unit
  • L may include a functional group used in the preparation of the conjugate at the terminal before it is formed into a conjugate.
  • the long-acting conjugate according to the present invention may be in a form in which Q and Z are linked through the functional group, but is not limited thereto.
  • the linker containing the ethylene glycol repeating unit may include two, or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.
  • the linker may be polyethylene glycol (PEG) represented by the following formula (3), but is not limited thereto:
  • the PEG moiety in the long-acting conjugate may include not only the -(CH 2 CH 2 O) n -structure, but also an oxygen atom intervening between the linking element and the -(CH 2 CH 2 O) n -, but this It is not limited.
  • the polyethylene glycol is a term that encompasses all forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.
  • the ethylene glycol repeating unit may be represented by, for example, [OCH 2 CH 2 ]n, and the value of n is a natural number, the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, such as the number
  • the average molecular weight may be set to be greater than 0 to about 100 kDa, but is not limited thereto.
  • the n value is a natural number and the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, for example, has a number average molecular weight of about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa , about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa,
  • both ends of the linker may bind to a thiol group, an amino group, a hydroxyl group of an immunoglobulin Fc fragment, and a thiol group, an amino group, an azide group, or a hydroxyl group of the peptide of Formula 1, It is not limited thereto.
  • the linker comprises a reactive group capable of binding to the immunoglobulin Fc and the peptide of Formula 1, respectively, at both ends, specifically, the thiol group of the cysteine of the immunoglobulin Fc fragment; an amino group located at the N-terminus, lysine, arginine, glutamine and/or histidine; and/or a thiol group of a cysteine of the peptide of Formula 1 which is bonded to a hydroxyl group located at the C-terminus; amino groups of lysine, arginine, glutamine and/or histidine; azide group of azidolysin; and/or a reactive group capable of bonding to a hydroxyl group, but is not limited thereto.
  • the reactive group of the linker may be at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.
  • a propion aldehyde group or a butyl aldehyde group may be exemplified as the aldehyde group, but the present invention is not limited thereto.
  • succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N -Hydroxysuccinimide, hydroxy succinimidyl or succinimidyl carbonate may be used, but not limited thereto.
  • the linker may be connected to the immunoglobulin Fc fragment Z and the peptide of general formula 1 Q through the above-described reactive group to be converted into a linker linker.
  • the final product resulting from reductive alkylation with aldehyde bonds is much more stable than those linked with amide bonds.
  • the aldehyde reactive group selectively reacts with the N-terminus at a low pH, and may form a covalent bond with a lysine residue at a high pH, for example, pH 9.0, but is not limited thereto.
  • the terminal reactive groups of the linker of the present invention may be the same as or different from each other.
  • the linker may have an aldehyde group reactive group at the end, and the linker may have an aldehyde group and a maleimide reactive group at the end, respectively, or may have an aldehyde group and a succinimide reactive group at the end, respectively, but is not limited thereto does not
  • it may have a maleimide group at one end and an aldehyde group, propionaldehyde group or butyraldehyde group at the other end.
  • it may have a succinimidyl group at one end and a propionaldehyde group or a butyl aldehyde group at the other end.
  • the hydroxyl group can be activated into the various reactive groups by a known chemical reaction, or a commercially available polyethylene glycol having a modified reactive group is used.
  • the conjugates of the invention can be prepared.
  • the reactive group of the linker may be linked to a cysteine residue of the peptide of Formula 1, more specifically, to a -SH group of cysteine, but is not limited thereto.
  • maleimide-PEG-aldehyde is used, the maleimide group is connected to the -SH group of the peptide of Formula 1 by a thioether bond, and the aldehyde group undergoes a reductive alkylation reaction with the -NH 2 group of the immunoglobulin Fc. may be connected through, but is not limited thereto, and this corresponds to one example.
  • the N-terminal amino group of the immunoglobulin Fc fragment is linked to an oxygen atom located at one end of PEG through a linker functional group having a structure of -CH 2 CH 2 CH 2 -, -PEG-O -CH 2 CH 2 CH 2 NH-
  • a linker functional group having a structure of -CH 2 CH 2 CH 2 -, -PEG-O -CH 2 CH 2 CH 2 NH-
  • one end of PEG through a thioether bond can form a structure linked to the sulfur atom located at the cysteine of the peptide of Formula 1 .
  • the above-mentioned thioether bond is may include the structure of
  • the reactive group of the linker may be linked to -NH 2 located at the N-terminus of the immunoglobulin Fc fragment, but this corresponds to one example.
  • the peptide of Formula 1 may be linked to a linker having a reactive group through the C-terminus, but this corresponds to one example.
  • C-terminus refers to the carboxy terminus of a peptide, and refers to a position capable of binding to a linker for the purpose of the present invention.
  • it may include all amino acid residues around the C-terminus as well as the most terminal amino acid residue at the C-terminus, and specifically includes the first to 20th amino acid residues from the most terminal. can, but is not limited thereto.
  • the conjugate of Formula 1 may have a structure of Formula 2 below.
  • Q is the peptide of Formula 1 described above;
  • Z is an immunoglobulin Fc fragment
  • n may be a natural number. In this case, the description of n is the same as described above.
  • the long-acting conjugate of Formula 2 has a structure in which the peptide Q of Formula 1 of SEQ ID NO: 47 and the immunoglobulin Fc fragment Z are covalently linked through an ethylene glycol repeat, each Q of Formula 2 In the succinimide ring, Z may be in a form connected to the oxypropylene group of formula (2).
  • n may be such that the average molecular weight of the [OCH 2 CH 2 ]n site in the peptide conjugate, such as the number average molecular weight, is 1 to 100 kDa, or 1 to 20 kDa or 10 kDa. However, it is not limited thereto.
  • Q of the peptide conjugate may be a peptide having activity on the glucagon receptor, the GLP-1 receptor and the GIP receptor.
  • the moiety at which Q is connected to the succinimide ring of Formula 2 may be the sulfur atom of the C-terminal cysteine of Q.
  • Z in Formula 1 or 2 is an immunoglobulin Fc fragment, and when referred to herein as an immunoglobulin Fc fragment, not only the native sequence obtained from papain digestion of immunoglobulin, but also derivatives and substitutions thereof, for example, one or more amino acid residues in the native sequence are deleted. , including modifications such as sequences that have been transformed by insertion, non-conservative or conservative substitution, or a combination thereof to be different from the native type.
  • the above derivatives, substituents and variants are premised on retaining the ability to bind FcRn.
  • part connected to the said oxypropylene group in Z is not specifically limited.
  • the moiety of Z connected to the oxypropylene group may be an N-terminal nitrogen or a nitrogen atom of a Z moiety (eg, epsilon nitrogen of lysine).
  • the moiety where Z is connected to the oxypropylene group of Formula 1 may be the N-terminal proline of Z, but is not limited thereto.
  • Z is a structure in which two polypeptide chains are connected by a disulfide bond, and may be a structure in which only one of the two chains is connected through a nitrogen atom, but is not limited thereto.
  • the linkage through the nitrogen atom may be connected through reductive amination to the epsilon amino atom or the N-terminal amino group of lysine, but is not limited thereto.
  • Reductive amination reaction means a reaction in which an amine group or an amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to form an amine and then forms an amine bond by a reduction reaction, It is an organic synthesis reaction well known in the art.
  • the Z may be connected through the nitrogen atom of the N-terminal proline, but is not limited thereto.
  • immunoglobulin Fc fragment refers to a heavy chain constant region excluding the heavy and light chain variable regions of an immunoglobulin.
  • the immunoglobulin Fc fragment may include a heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3) portion, and more specifically, the hinge region (means all or part of the hinge region). .) may be further included.
  • the immunoglobulin Fc fragment is one component constituting a moiety of the peptide conjugate of Formula 1 of the present invention, and specifically, may correspond to Z in Formula 1 above.
  • the immunoglobulin Fc fragment may include a hinge portion in the heavy chain constant region, but is not limited thereto.
  • the immunoglobulin Fc fragment may include a specific hinge sequence at the N-terminus.
  • flankinge sequence refers to a region that is located on a heavy chain and forms a dimer of an immunoglobulin Fc fragment through an inter disulfide bond.
  • the hinge sequence may be mutated to have only one cysteine residue by deleting a part of the hinge sequence having the following amino acid sequence, but is not limited thereto:
  • the hinge sequence may include only one cysteine residue by deleting the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO: 55.
  • the hinge sequence of the present invention may be composed of 3 to 12 amino acids, including only one cysteine residue, but is not limited thereto.
  • the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 56), Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 57), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 58), Glu- Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 59), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 60), Glu-Ser-Lys- Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 61), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO:
  • the hinge sequence may include the amino acid sequence of SEQ ID NO: 74 (Ser-Cys-Pro) or SEQ ID NO: 76
  • the immunoglobulin Fc fragment of the present invention may be in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence, and in the long-acting conjugate of Formula 1 of the present invention, one end of the linker is a dimer It may be in a form linked to one chain of the immunoglobulin Fc fragment, but is not limited thereto.
  • N-terminus refers to the amino terminus of a protein or polypeptide, and 1, 2, 3, 4, 5, 6, It may include up to 7, 8, 9, or 10 or more amino acids.
  • the immunoglobulin Fc fragment of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.
  • part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region except for only the heavy and light chain variable regions of immunoglobulin 1 (CL1) may be an extended Fc region. It may also be a region in which some fairly long amino acid sequences corresponding to CH2 and/or CH3 have been removed.
  • the immunoglobulin Fc fragment of the present invention comprises 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or two or more of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), 6) heavy chain constant region each domain and the light chain constant region may be a dimer .
  • the immunoglobulin Fc fragment may be in a dimeric form, and one molecule of the peptide of Formula 1 may be covalently linked to one Fc region in the dimeric form, In this case, the immunoglobulin Fc and the peptide of Formula 1 may be linked to each other by a linker containing an ethylene glycol repeating unit.
  • the immunoglobulin Fc and the peptide of Formula 1 may be linked to each other by a linker containing an ethylene glycol repeating unit.
  • the immunoglobulin Fc fragment of the present invention includes a native amino acid sequence as well as a sequence derivative thereof.
  • An amino acid sequence derivative means that one or more amino acid residues in a native amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for binding may be used as suitable sites for modification.
  • various types of derivatives are possible, such as a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus of native Fc are removed, or a methionine residue may be added to the N-terminus of native Fc do.
  • the complement binding site eg, the C1q binding site
  • the ADCC antibody dependent cell mediated cytotoxicity
  • the above-described Fc derivative may exhibit biological activity equivalent to that of the Fc fragment of the present invention, and may have increased structural stability against heat, pH, etc. of the Fc fragment.
  • the Fc fragment may be obtained from a native type isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
  • the method of obtaining from the native type may be a method of obtaining whole immunoglobulins by treatment with proteolytic enzymes after isolating whole immunoglobulins from a living body of a human or animal. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2.
  • Fc or pF'c may be separated using size-exclusion chromatography or the like.
  • the human-derived Fc fragment is a recombinant immunoglobulin Fc fragment obtained from a microorganism.
  • the immunoglobulin Fc fragment may be in the form of a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed.
  • Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used for the increase or decrease or removal of such immunoglobulin Fc sugar chains.
  • the immunoglobulin Fc fragment from which the sugar chain has been removed from Fc significantly lowers the binding force to complement (c1q) and reduces or eliminates antibody-dependent cytotoxicity or complement-dependent cytotoxicity, so that unnecessary immune responses are not induced in vivo does not
  • a form more suitable for the original purpose as a drug carrier will be an immunoglobulin Fc fragment in which sugar chains are removed or non-glycosylated.
  • deglycosylation refers to an Fc fragment from which sugars are removed with an enzyme
  • aglycosylation refers to an Fc fragment that is not glycosylated by production in prokaryotes, in a more specific embodiment, in E. coli. .
  • the immunoglobulin Fc fragment may be of human or animal origin, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in a more specific embodiment, it is of human origin.
  • the immunoglobulin Fc fragment may be an Fc fragment derived from IgG, IgA, IgD, IgE, or IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in a more specific embodiment is derived from IgG, which is known to enhance the half-life of ligand binding proteins. In an even more specific embodiment, the immunoglobulin Fc fragment is an IgG4 Fc fragment, and in the most specific embodiment, the immunoglobulin Fc fragment is a non-glycosylated Fc fragment derived from human IgG4, but is not limited thereto.
  • the immunoglobulin Fc fragment is a fragment of human IgG4 Fc, and is a homologue in which two monomers are linked through a disulfide bond (inter-chain form) between cysteine, amino acid 3 of each monomer. It may be in the form of a dimer, wherein each monomer of the homodimer is independently an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, i.e., two internal and/or may have disulfide bonds (in an intra-chain form).
  • the number of amino acids of each monomer may consist of 221 amino acids, and the amino acids forming the homodimer may consist of a total of 442 amino acids, but is not limited thereto.
  • two monomers having the amino acid sequence of SEQ ID NO: 76 (consisting of 221 amino acids) form a homodimer through a disulfide bond between cysteine, the 3rd amino acid of each monomer, and the homodimer
  • the monomers of may each independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, but is not limited thereto.
  • Z of Formula 1 may include a monomer having an amino acid sequence of SEQ ID NO: 76, and Z may be a homodimer of a monomer having an amino acid sequence of SEQ ID NO: 76, but is not limited thereto.
  • the immunoglobulin Fc fragment may be a homodimer comprising the amino acid sequence of SEQ ID NO: 75 (consisting of 442 amino acids), but is not limited thereto.
  • dimer or multimers when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc fragment of the same origin forms a bond with a single-chain polypeptide of a different origin. That is, dimers or multimers can be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.
  • hybrid is a term that means that sequences corresponding to immunoglobulin Fc fragments of two or more different origins exist in a single-chain immunoglobulin constant region.
  • various types of hybrids are possible. That is, a hybrid of domains consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge.
  • IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and in the present invention, a combination thereof or hybridization thereof is also possible. Specifically, it is an IgG2 and IgG4 subclass, and most specifically, an Fc fragment of IgG4 having little effector function such as complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the liquid formulation may be for the prevention or treatment of metabolic syndrome.
  • prevention refers to any act of inhibiting or delaying the onset of metabolic syndrome by administration of the conjugate or a formulation containing the same
  • treatment is the administration of the conjugate or a preparation containing the same to treat metabolic syndrome. It means any action that improves or benefits symptoms.
  • the term "administration” means introducing a predetermined substance to a patient by any suitable method, and the route of administration of the composition is not particularly limited thereto, but any general route through which the composition can reach an in vivo target It may be administered through, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.
  • routes of administration of the composition is not particularly limited thereto, but any general route through which the composition can reach an in vivo target It may be administered through, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.
  • Another aspect embodying the present invention provides a method for preparing the liquid formulation.
  • the production method is (a) a glucagon receptor, a GLP-1 receptor, and a peptide and immunoglobulin Fc fragment having activity on the GIP receptor are linked to each other, glucagon receptor, GLP-1 receptor, and activity on the GIP receptor It may include the step of mixing with each other a long-acting conjugate of a peptide with
  • the stabilizer may further include one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids, but is not particularly limited thereto.
  • the long-acting conjugate, buffer, sugar alcohol, sugar, or a combination thereof, isotonic agent, nonionic surfactant, amino acid, and stabilizer are the same as described above.
  • Another embodiment embodying the present invention provides a method for preventing or treating metabolic syndrome, comprising administering the liquid formulation to an individual in need thereof.
  • liquid formulation, metabolic syndrome, prevention, and treatment are the same as described above.
  • the subject is an individual in need of administration of the formulation of the present invention, and includes, without limitation, an individual that can be treated with the liquid formulation of the present invention, and specifically includes humans or mammals including mice and livestock.
  • the treatment method of the present invention may include administering a liquid formulation in a pharmaceutically effective amount.
  • a suitable total daily amount may be determined by a treating physician within the scope of sound medical judgment, and may be administered once or divided into several doses.
  • a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.
  • Another aspect embodying the present invention is the use of the liquid formulation in the manufacture of a medicament for the prevention or treatment of metabolic syndrome.
  • liquid formulation, metabolic syndrome, prevention, and treatment are the same as described above.
  • Another aspect embodying the present invention is the use of the liquid formulation for use in the prevention or treatment of metabolic syndrome.
  • liquid formulation, metabolic syndrome, prevention, and treatment are the same as described above.
  • a long-acting conjugate of glucagon, GLP-1 and triple activator of GIP was prepared by the following method.
  • a triple activator exhibiting activity on all GLP-1 receptors, GIP receptors and glucagon receptors was prepared, and its sequences are shown in Table 1 below.
  • an amino acid denoted by X is a non-natural amino acid Aib (aminoisobutyric acid), and an underlined amino acid means that the underlined amino acids form a ring with each other.
  • Aib amino acid denoted by X
  • underlined amino acid means that the underlined amino acids form a ring with each other.
  • CA means 4-imidazoacetyl
  • Y means tyrosine.
  • Each of the above cell lines was transformed to express human GLP-1 receptor, human GCG receptor and human GIP receptor genes in Chinese hamster ovary (CHO), respectively, and is suitable for measuring the activities of GLP-1, GCG and GIP. Therefore, the activity for each part was measured using each transformed cell line.
  • human GLP-1 was serially diluted from 50 nM to 0.000048 nM by 4 times, and the triple activator prepared in 1-1 was diluted from 400 nM to 4 times each. Serial dilutions to 0.00038 nM.
  • the culture medium was removed from the cultured CHO cells expressing the human GLP-1 receptor, 5 ⁇ l of each serially diluted substance was added to the cells, and then 5 ⁇ l of a buffer containing cAMP antibody was added for 15 minutes. during incubation at room temperature. Then, 10 ⁇ l of a detection mix containing a cell lysis buffer was added to lyse the cells and reacted at room temperature for 90 minutes.
  • the cell lysate was applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value through the accumulated cAMP, and then compared with each other. Relative titers compared to human GLP-1 are shown in Table 2 below.
  • human GCG was serially diluted from 50 nM to 0.000048 nM in 4 folds, and the triple activator prepared in 1-1 was continuously diluted from 400 nM to 0.00038 nM in 4 folds.
  • the culture medium was removed from the cultured CHO cells expressing the human GCG receptor, 5 ⁇ l of each serially diluted substance was added to the cells, and 5 ⁇ l of a buffer containing cAMP antibody was added thereto, followed by room temperature for 15 minutes. cultured in Then, 10 ⁇ l of a detection mix containing a cell lysis buffer was added to lyse the cells and reacted at room temperature for 90 minutes.
  • the cell lysate was applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value through the accumulated cAMP, and then compared with each other. Relative titers compared to human GCG are shown in Table 2 below.
  • human GIP was serially diluted from 50 nM to 0.000048 nM in 4 folds, and the triple activator prepared in 1-1 was continuously diluted from 400 nM to 0.00038 nM in 4 folds.
  • the culture medium was removed from the cultured CHO cells expressing the human GIP receptor, 5 ⁇ l of each serially diluted substance was added to the cells, and 5 ⁇ l of a buffer containing cAMP antibody was added thereto, followed by room temperature for 15 minutes. cultured in Then, 10 ⁇ l of a detection mix containing a cell lysis buffer was added to lyse the cells and reacted at room temperature for 90 minutes.
  • the cell lysate was applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC 50 value through the accumulated cAMP, and then compared with each other. Relative titers compared to human GIP are shown in Table 2 below.
  • a long-acting conjugate was prepared using the peptide of SEQ ID NO: 9 as a representative triple activator.
  • a linearly modified polyethylene glycol maleimide-PEG-aldehyde (NOF, Japan) with the cysteine residue of the triple activator
  • the triple activator was pegylated to the maleimide end of maleimide-PEG-aldehyde.
  • the molar ratio of the triple activator to maleimide-PEG-aldehyde was 1:1 to 3, and the protein concentration was 1 to 5 mg/ml, and the reaction was carried out at low temperature for 0.5 to 3 hours. At this time, the reaction was carried out in an environment in which 20 to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After completion of the reaction, the reaction solution was applied to SP Sepharose HP (GE healthcare, USA) to purify the tri-activator mono-pegylated to cysteine.
  • SP Sepharose HP GE healthcare, USA
  • the immunoglobulin Fc fragment was published internationally using an immunoglobulin Fc fragment (49.8 kDa, two chains of SEQ ID NO: 76 linked by disulfide bonds) having a hinge region of the Pro-Ser-Cys-Pro sequence at the N-terminus. It was prepared by the method described in patent WO2007/021129.
  • the purified mono-pegylated triple activator and immunoglobulin Fc are mixed with a molar ratio of 1:1 to 5 and a protein concentration of 10 to 50 mg/mL at 4 to 8°C for 12 to 18 hours. reacted.
  • the reaction was carried out in an environment in which 10 to 50 mM sodium cyanoborohydride and 10 to 30% isopropanol as reducing agents were added to 100 mM potassium phosphate buffer (pH 6.0).
  • reaction solution was applied to the butyl sepharose FF purification column (GE healthcare, USA) and the Source ISO purification column (GE healthcare, USA), and the mono-pegylated tri-activator of the aldehyde side polyethylene A long-acting conjugate of a triple activator in which the glycol terminus is linked to the N-terminal proline nitrogen of one of the two chains of the immunoglobulin Fc homodimer was purified.
  • glucagon, GLP-1 and GIP triple activators and immunoglobulin Fc fragments are linked via PEG was designated as a 'long-acting conjugate of glucagon, GLP-1 and GIP triple activator'.
  • Example 1 Evaluation of stability of long-acting conjugates of glucagon, GLP-1 and GIP triple activators according to pH
  • the stability of long-acting conjugates of glucagon, GLP-1 and GIP triple activators was compared under various pHs based on a liquid formulation consisting of a buffer substance, mannitol as a sugar alcohol, polysorbate 20 as a surfactant, and methionine.
  • a formulation having a pH of 4.5 was used as a comparative example.
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation of the composition shown in Table 3 (the concentration of the long-acting conjugate was 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography ( Stability was analyzed using Ion Exchange High Performance Liquid Chromatography (IE-HPLC) and Reverse Phase-High Performance Liquid Chromatography (RP-HPLC).
  • IE-HPLC Ion Exchange High Performance Liquid Chromatography
  • RP-HPLC Reverse Phase-High Performance Liquid Chromatography
  • IE-HPLC (%) and RP-HPLC (%) in Table 4 are percentage values of the ratio (Area% / Start Area%) obtained by dividing the area % value at the measurement time by the initial area % value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • formulation (#1) having a composition of sodium citrate, pH 5.0, and formulation (#2) having a composition of sodium citrate, pH 5.5 showed high stability at 25°C for 6 weeks.
  • the formulation having a pH 6.0 composition (#3) and a formulation having a pH 6.5 composition (#4) also showed stability for 6 weeks.
  • the composition of pH 4.5 which is a comparative example, it was confirmed that precipitation occurred at 6 weeks.
  • Example 2 Evaluation of stability of long-acting conjugates of glucagon, GLP-1 and GIP triple activators according to sugar or sugar alcohol type
  • monosaccharides such as mannose, glucose, fucose and xylose and lactose
  • polysaccharides such as maltose, sucrose, sorbitol, raffinose and dextran.
  • the stability of the long-acting conjugate of glucagon, GLP-1 and GIP triple activators according to mannitol, sucrose, and sorbitol confirmed in Example 1 was compared.
  • the concentration of mannitol, sucrose and sorbitol was considered the maximum allowable range recommended by the commercial formulation and the licensing agency.
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation having the composition shown in Table 5 (the concentration of the long-acting conjugate was 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography and reversed phase Analyzed using chromatography.
  • IE-HPLC (%) and RP-HPLC (%) in Table 6 are percentage values of the ratio (Area% / Start Area%) obtained by dividing the area % value at the measurement time by the initial area % value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • mannitol, sorbitol, and sucrose which are sugars or sugar alcohols that may be included in order to increase the storage stability of the long-acting conjugate of glucagon, GLP-1 and GIP triple activator, respectively, 5%, 5%, and When included at 8%, similar stability was shown.
  • Example 3 Evaluation of stability of long-acting conjugates of glucagon, GLP-1 and GIP triple activators according to sugar or sugar alcohol concentration
  • Glucagon, GLP-1 and GIP triple according to sugar or sugar alcohol concentration based on the composition (sodium citrate, pH 5.5, mannitol, polysorbate 20, and methionine) of the liquid formulation confirmed in Example 1 or Example 2
  • concentration of the isotonic agent added together was selected in consideration of the acceptable range of commercially available formulations and normal plasma osmotic pressure.
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation having the composition shown in Table 7 (the concentration of the long-acting conjugate was 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography and reverse phase Analyzed using chromatography.
  • IE-HPLC (%) and RP-HPLC (%) in Table 8 are percentage values of the ratio (Area% / Start Area%) obtained by dividing the area % value at the measurement time by the initial area % value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • Example 4 Stability evaluation of long-acting conjugates of glucagon, GLP-1, and triple activators of GIP according to the type of buffer substance
  • the stability of the long-acting conjugate of glucagon, GLP-1 and GIP triple activator was compared according to the type of buffer material. .
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation with the composition shown in Table 9 (the concentration of the long-acting conjugate was 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography and reverse phase Analyzed using chromatography.
  • IE-HPLC (%) and RP-HPLC (%) in Table 10 are the percentage values of the ratio (Area% / Start Area%) of the area percentage value at the measurement time divided by the initial area percentage value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • Example 5 Evaluation of stability of long-acting conjugates of glucagon, GLP-1 and GIP triple activators according to types of nonionic surfactants
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation having the composition shown in Table 11 (the concentration of the long-acting conjugate was 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography and reverse phase Analyzed using chromatography.
  • IE-HPLC (%) and RP-HPLC (%) in Table 12 are the percentage values of the ratio (Area% / Start Area%) of the area percentage value at the measurement time divided by the initial area percentage value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • Example 6 Evaluation of stability of long-acting glucagon, GLP-1 and GIP triple activator conjugates with or without nonionic surfactant and amino acids
  • the stability of the long-acting conjugate of glucagon, GLP-1 and GIP triple activator was compared when the liquid formulation contained or did not contain a nonionic surfactant or an amino acid stabilizer.
  • the long-acting conjugate of the triple activator obtained in Preparation Example above was prepared as a liquid formulation having the composition shown in Table 13 (long-acting conjugate concentration is 183.79 nmol/mL) and stored at 25° C. for 6 weeks, followed by ion exchange chromatography and reverse phase Analyzed using chromatography.
  • IE-HPLC (%) and RP-HPLC (%) in Table 14 are percentage values of the ratio (Area% / Start Area%) obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the preservation test. Residual ratio from the initial concentration (183.79 nmol/mL concentration) of the long-acting conjugate is shown.
  • Example 7 Evaluation of stability of long-acting glucagon, GLP-1 and GIP triple activator conjugates according to concentration

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